Female reproductive functioning requires the precise temporal organization of numerous neuroendocrine events by a master circadian brain clock located in the suprachiasmatic nucleus (SCN). Across species, including humans, disruptions to circadian timing result in pronounced deficits in ovulation and fecundity. Our investigations focus on the circadian control of two key neuropeptides with opposing roles, gonadotropin-inhibitory hormone (GnIH, also known as RFRP-3) and kisspeptin. Across mammalian species, GnIH markedly inhibits the secretion of gonadotropin-releasing hormone (GnRH) and pituitary gonadotropin secretion, whereas kisspeptin is a pronounced stimulator of the GnRH neuronal network. Despite the well- established roles for both GnIH and kisspeptin in mammalian reproduction, as well as the knowledge that the circadian timing system is a crucial regulator of the female reproductive axis, the specific neurochemical pathways underlying these interactions are not well understood. The present proposal explores how a hierarchy of circadian oscillators interacts with the GnIH and kisspeptin signaling pathways to regulate GnRH secretion and the preovulatory GnRH/luteinizing hormone (LH) surge. Our work to date indicates that the SCN projects monosynaptically to the GnIH and kisspeptin systems to coordinate their activational states appropriately to allow for initiation of the GnRH/LH surge and ovulation. Additionally, our recent in vitro and in vivo findings point to a novel role for autonomous circadian clocks, operating in GnRH cells, in mediating daily responsiveness of the HPG axis to upstream neurochemical signaling, including kisspeptin. The present proposal combines system, circuit, molecular/genetic, and pharmacological approaches to investigate the interactions among the GnIH, kisspeptin, and circadian systems by exploring: 1) the specific neural loci at which kisspeptin and GnIH interact to regulate GnRH secretion, 2) the neurochemical means by which the SCN coordinates the timed secretion of kisspeptin and GnIH, and 3) the functional implications of these interactions for the GnRH/LH surge and ovulation. The proposed work addresses a classic question in regulatory biology, and has the potential for substantial translational impact in the development of safe/effective contraception, as well as the treatment of a host of reproductive disorders in humans, including precocious and delayed puberty, infertility, and polycystic ovarian syndrome. PUBLIC HEALTH RELEVANCE: Converging lines of evidence implicate a critical role for circadian timing in successful female reproduction across mammalian species, including humans. Women with irregular work or sleep cycles, for example, experience abnormal menstrual cycles, difficultly becoming pregnant, and an increased rate of spontaneous abortions. Given the necessity of proper hormonal timing in reproductive health, our studies examine the role of circadian brain clocks in the coordination of key positive and negative neurochemical regulatory systems critical for ovulation and reproductive success.